The present invention relates generally to a recombinant viral expression system. More particularly, the present invention relates to a highly efficient, recombinant adenovirus expression system capable of expressing a heterologous gene(s) in a host mammalian cell.
The human adenovirus-5 (Ad5) genome consists of a double-stranded linear DNA molecule of 36 kilo-basepair (bp) (Ginsberg, 1984). The virus replication cycle has two phases: an early phase, during which 4 transcription units E1, E2, E3, and E4 are expressed, and a late phase occurring after the onset of viral DNA synthesis when late transcripts are expressed from the major late promoter (MLP). These late messages encode most of the viral structural proteins. E1, E2 and E4 gene products of human adenoviruses are involved in transcriptional activation, cell transformation, and viral DNA replication as well as other viral functions, and are essential for viral growth (Grand, 1987, Biochem. J., vol. 241, pp. 25-38; and Nevins, 1987, Microbiol. Rev., vol. 51, pp. 419-430). In contrast, E3 gene products are not required for viral replication in cultured cells (Ginsberg et al., 1989,), but appear to be involved in evading immune surveillance in vivo (Anderson et al., 1985, Cell, vol. 43, pp. 215-222; Burgert et al., 1985, Cell, vol. 41, pp. 987-997; Burgert et al., 1987, EMBO J., vol. 6, pp. 2019-2026; Carlin et al., 1989, Cell, vol. 57, pp. 135-144; Gooding and Wold, 1990, Crit. Rev. Immunol., vol. 10, pp. 53-71; Gooding et al., 1988, Cell, vol. 53, pp. 341-346; Horton et al., 1990, J. Virol., vol. 64, pp. 1250-1255; Tollefson et al., 1991, J. Virol., vol. 65, pp. 3095-3105; Wold and Gooding, 1989, Mol. Biol. Med., vol. 6, pp. 433-452; and Wold and Gooding, 1991, Virology, vol. 184, pp. 1-8).
E1 and E3 and a site upstream of E4 have been utilized as sites for insertion of foreign DNA sequences in the generation of recombinant adenoviruses (Berkner et al., 1984, Nuc. Acids. Res., vol. 12, pp. 1925-1941; Chanda et al., 1990, Virology, vol. 175, pp. 535-547; Haj-Ahmad et al., 1986, J. Virol., vol. 57, pp. 267-274; and Saito et al., 1985, J. Virol., vol. 54, pp. 711-719). Since the upper size limit for DNA molecules that can be packaged into adenovirus particles is approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987, EMBO J., vol. 6, pp. 1733-1739), only about 2 kb of extra DNA can be inserted without compensating deletions of viral DNA. Although E1 is essential for virus replication in cell culture, foreign DNA can be substituted for E1 sequences when the virus is grown in 293 cells which are transformed by adenovirus-5 DNA and constitutively express E1 (Graham et al., 1977, J. Gen. Virol., vol. 36, pp. 59-72). Several vectors having 1.9 kb deleted from E3 of adenovirus-5 have been constructed without interfering with virus replication in cell culture (Graham et al., 1992, Vaccines; New Approaches to Immunological Problems, R. W. Ellis (Ed.), Butterworth-Heinemann, Boston, Mass., pp. 364-390). Such vectors allow for insertion of up to 4 kb of foreign DNA. Recombinant adenoviruses containing inserts in E3 replicate in all adenovirus-permissive cell lines and may be suitable as live recombinant viral vaccines since a number of adenovirus vectors containing E3 inserts have been shown to express foreign genes efficiently both in vitro and in vivo (Berkner, 1988: Chanda et al., 1990; Dewar et al., 1989, J. Virol., vol. 63, pp. 129-136; Graham, 1990, Trends Biotechnol., vol. 8, pp. 85-87; Graham et al., 1992; Johnson et al., 1988, Virology, vol. 164, pp. 1-14; Lubeck et al., 1989, Proc. Natl. Acad. Sci. USA, vol. 86, pp. 6763-6767; McDermott et al. 1989, Virology, vol. 169, pp. 244-247; Morin et al., 1987, Proc. Natl. Acad. Sci. USA, vol. 84, pp. 4626-4630; Prevec et al., 1989, J. Gen. Virol., vol. 70, pp. 429-434; Prevec et al., 1990, J. Inf. Dis., vol. 161, pp. 27-30; Schneider et al., 1989, J. Gen. Virol. , vol. 70, pp. 417-427; Vernon et al., 1991, J. Gen. Virol., vol. 72, pp. 1243-1251; and Yuasa et al., 1991, J. Gen. Virol., vol. 72, pp. 1927-1934).
Adenoviruses are good mammalian cell expression vectors with potential utility as live recombinant vaccines, in gene therapy, or for high level protein production in mammalian cells.
Adenovirus expression vectors have been in use for the past decade (Thummel et al., 1981, Cell, vol. 23, pp. 825-836; Berkner et al., 1984, Nucleic Acids Res., vol. 12, pp. 1925-1941; and for a review see Grunhaus et al., 1992, Seminars in Virology 3, pp. 237-252), and more recently exploited for the purpose of gene therapy (Herz et al., 1993, Proc. Natl. Acad. Sci. U.S.A., vol. 90, pp. 2812-2816; Rosenfeld et al., 1991, Science, vol. 252, pp. 431-434; and Rosenfeld et al., 1992, Cell, vol. 68, pp. 143-155). Features of adenovirus based expression vectors which make them attractive to gene therapy applications include very efficient uptake into cells which contain the appropriate adenovirus receptor and uptake pathway, and the ability to carry up to 7.5 kb of foreign DNA. Adenovirus vectors allow a reporter gene to be under the control of tissue specific promoter elements (Friedman et al., 1986, Mol. Cell. Biol., vol. 6, pp. 3791-3797; and Babiss et al., 1986, Mol. Cell. Biol., vol. 6, pp. 3798-3806) as well as a variety of viral and mammalian constitutive promoter elements (Mittal et al., 1993, Virus Research, vol. 28, pp. 67-90).
One such example of an adenovirus-based vector system is described in Mittal et al., 1993, Virus Research, vol. 28, pp. 67-90. The authors here describe a helper-independent adenovirus type 5-luciferase recombinant containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted into the early region 3 (E3) of the adenovirus-5 genome. A plasmid containing the luciferase gene and SV40 regulatory sequences in the E3 region was co-transfected with a plasmid containing the adenovirus-5 d1309 genome in circular form. Upon transfection of 293 cells, virus progeny produced by in vivo recombination between the two plasmids resulted in rescue of the adenovirus type 5-luciferase recombinant (i.e., E3 insert in Adenovirus-5 genome).
Gomez-Foix et al., 1992, J. Biol. Chemistry, vol. 267, no. 35, pp. 25129-25134, discloses adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes in culture. The preparation of a recombinant adenovirus containing the cDNA encoding rabbit muscle glycogen phosphorylase is described whereby the cytomegalovirus (CMV) early gene promoter/enhancer, pUC 18 polylinker, fragment of the SV40 genome that includes the small T-antigen intron and the polyadenylation signal, and cDNA that includes all of the protein coding region of the rabbit muscle glycogen phosphorylase, was inserted into vector pAC. The resulting plasmid was co-transfected into 293 cells with plasmid pJM17, which encodes a full-length adenovirus-5 genome. Homologous recombination between the recombinant plasmids in 293 cells generated a genome of packageable size in which the adenovirus early region 1 was replaced by the cloned chimeric gene encoding rabbit muscle glycogen phosphorylase.
Roessler et al., 1993, J. Clin. Invest., discloses using a recombinant adenoviral vector for the expression of the gene for Escherichia coli beta-galactosidase within synovium tissue. Replication defective adenoviral vectors are deleted of sequences spanning E1A, E1B and a portion of the E3 region, impairing the ability of this virus to replicate or transform nonpermissive cells. The early enhancer/promoter of the cytomegalovirus (CMV) was inserted into this vector to drive transcription of lacZ with a SV40 polyadenylation sequence cloned downstream from this reporter.
Yang et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 9480-9484, discloses the expression of cystic fibrosis transmembrane conductance regulator (CFTR) by adenovirus-mediated gene transfer. The recombinant adenoviruses were produced by homologous recombination of two vectors which contain the following relevant sequences: 5xe2x80x2 ITR of adenovirus-5 spanning 0-1 map units; Tha I-SnaBI fragment of the immediate-early gene of cytomegalovirus; promoter from the chicken xcex2-actin gene spanning Xho I at nucleotide (nt) xe2x88x92275 to Mbo I at nt +1; human CFTR cDNA containing 60 nt of 5xe2x80x2 untranslated sequence, the entire coding sequence, and 80 nt of 3xe2x80x2 untranslated sequence; simian virus 40 late gene polyadenylation signal; 9.2-16.1 map units of adenovirus-5; and plasmid sequences.
Herz et al., 1993, Proc. Natl. Acad. Sci. USA, vol. 90, pp. 2812-2816, discloses the use of adenovirus-mediated gene transfer to transiently elicit production of low density lipoprotein (LDL) receptors in mice. Recombinant adenoviruses containing: 1) cDNA encoding the human LDL receptor (AdCMV-LDLR)(CMV,cytomegalovirus); 2)xcex2-galactosidase (AdCMV-xcex2gal); and firefly luciferase (AdCMV-Luc), were prepared using co-transfection of the appropriate plasmids in 293 cells.
Rosenfeld et al., 1991, Science, vol. 252, pp. 431-434, discloses adenovirus-mediated transfer of recombinant xcex11-antitrypsin gene to the lung epithelium cells of the cotton rat respiratory tract in vivo. The adenoviral vector contained an adenovirus major late promoter and a recombinant human xcex11-antitrypsin gene.
Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, vol. 89, pp. 2581-2584, discloses a recombinant adenovirus containing the xcex2-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus directed expression of the xcex2-galactosidase in myotubes in vivo.
Problems associated with adenovirus infection, particularly those associated with repression of host cell mRNA translation and shutdown of host normal mRNA production (Babich et al., 1983, Mol. Cell. Biol., vol. 3, pp. 1212-1221; Beltz et al., 1979, J. Mol. Biol., vol. 131, pp. 353-373; Schneider et al., 1987, Annu. Rev. Biochem., vol. 56, pp. 317-332) have been addressed by using defective adenovirus vectors which are based on mutations in the dominant regulatory region, E1 (Harrison et al., 1977, Virology, vol. 77, pp. 319-329; Jones et al., 1979, Cell, vol. 17, pp. 583-689). In addition, conventional adenovirus vector systems typically require high cell exposure (e.g., MOI""s in excess of 500 PFU/cell) for expression of the desired gene, which is detrimental to the cells because of cytopathic effects from exposure. Therefore, a need exists for an adenovirus-mediated expression vector which can infect cells at low doses, yet can exhibit maximum expression of a gene in the cell.
Moreover, although adenovirus-based vectors for gene expression have been successfully employed with a number of mammalian and viral genes (for review, see Mulligan, R. C., 1993, Science, vol. 260, pp. 926-932), they have not apparently been used to express any member of the guanine nucleotide-binding protein coupled receptors (GPCR) family, such as the pituitary thyrotropin-releasing hormone (TRH-R)(Straub et al., 1990, Proc. Natl. Acad. Sci U.S.A., vol. 87, pp. 9514-9518; Yamada et al., 1992, Biochem. Biophys. Res. Commun., vol. 184, pp. 367-372; Zhao et al., 1992, Endocrinology, vol. 130, pp. 3529-3536; de la Pena et al., 1992, Biochem. J., vol. 284, pp. 891-899). Seven transmembrane-spanning GPCRs comprise a large family of cell surface regulatory proteins (Dohlman et al., 1991, Annu. Rev. Biochem., vol. 60, pp. 653-688). When studying the molecular details of receptor biology in mammalian cells, expression of wild type and mutant receptors is usually accomplished by gene transfer by one of several transfection procedures.
Assays using 1) a cell system that permits intracellular replication of the plasmid vector during transient expression studies; or 2) transfectants that stably express the receptor of interest, provide useful, but, limited receptor expression. Where transfections yield low levels of receptor expression, or where the range of cell types that can be transfected is restricted, studies of these receptors is limited. Adenovirus-mediated gene transfer could be employed as an alternative strategy to plasmid based receptor expression vectors. A significant advantage of using adenovirus-mediated gene transfer is the wide variety of cells which are susceptible to infection by adenovirus. This should permit study of TRH-R biology in a variety of mammalian cell types, including those not amenable to transfection techniques.
Furthermore, the analysis of elements involved in cardiac myocyte gene regulation would be greatly facilitated by a simple and efficient method of adenovirus-mediated gene transfer. Because there are no permanent cardiac myocyte cell lines, the majority of cardiac myocyte gene expression studies have been carried out using transient gene transfer techniques into primary cultures of fetal and neonatal cardiocytes (Gustafson et al., 1987, Proc. Natl. Acad. Sci. U.S.A., vol. 84, pp. 3122-3126). Although useful, this methodology has many limitations, including relatively low efficiency as well as being restricted to fetal and neonatal stages of development since transient transfection of adult cardiac myocytes has not been reported.
As an alternative, in vitro studies of cardiac myocyte gene regulation and gene transfer have been successfully carried out in transgenic (Rindt et al., 1993, J. Biol. Chem., vol. 268, pp. 5332-5338; and Subramanian et al., 1991, J. Biol. Chem., vol. 266, pp. 24613-24620). However, the generation of transgenic mouse lines is both costly and extremely time consuming.
A second approach to cardiac gene transfer in vitro has relied on injecting plasmid DNA into the myocardium and measuring reporter gene expression in the cells which have successfully taken up sufficient quantities of DNA (Kitsis et al., 1991, Proc. Natl. Acad. Sci. U.S.A., vol. 88, pp. 4138-4142; Lin et al., 1990, Circulation, vol. 82, pp. 2217-2221; and Ascadi et al., 1991, The New Biologist, vol. 3, pp. 71-81). The problem associated with direct DNA injection is its relative inefficiency as only approximately 0.02% of the myocytes in the adult rat heart take up and express injected DNA (Kitsis et al., 1993, in Methods in Molecular Genetics, ed. Adolph, K. W., Academic Press, Inc., New York, Vol. 1, pp. 374-392).
A recent report demonstrated efficient gene transfer into adult rat cardiocytes in vitro (Kirshenbaum et al., 1993, J. Clin. Invest., vol. 92, pp. 381-387). In addition, recent studies using adenovirus vectors introduced intravenously into both rats and mice, indicate that the virus will infect a wide variety of tissue types, including mouse skeletal and cardiac muscle (Quantin et al., 1992, Proc. Natl. Acad. Sci. U.S.A., vol. 89, pp. 2581-2584; and Strattford-Perricaudet et al., 1992, J. Clin. Invest., vol. 90, pp. 626-630). However, little quantitative data is available concerning expression of adenovirus-mediated gene transfer in vivo. Therefor, a need exists for an adenovirus-mediated gene transfer vector system which would function effectively with primary cultures of cardiac myocytes and one which would also have application in vitro.
The primary object of the present invention is to provide an adenovirus-based expression system capable of expressing a heterologous gene(s) in a host mammalian cell.
The present invention provides a novel, highly efficient, recombinant adenovirus expression system for expression of a heterologous gene(s) and/or gene product(s) in a mammalian cell. The recombinant adenovirus expression system of the invention was produced via homologous recombination between the novel vector of the invention co-transfected with the large fragment of the adenovirus-5 genome in 293 cells.
In accordance with the present invention, the novel expression vector is preferably a plasmid vector. The plasmid vector of the invention can be used as a generic vector, that is, for the expression of any number of selected heterologous gene(s). The generic plasmid vector is designated pAdCMV-HS-Vector. The plasmid vector described herein can itself be transfected into a mammalian cell for the expression of any number of gene(s) and/or production of a gene product(s), depending on the heterologous gene(s) cloned into the plasmid vector. Alternatively, the plasmid vector can be converted into the recombinant adenovirus of the invention. Examples of various uses of the plasmid vector are described in the various embodiments disclosed herein.
In one embodiment of the invention, the plasmid vector includes at least one cDNA insertion site, i.e., restriction site(s) for cloning a selected heterologous gene(s). Positioned upstream of the gene insertion site(s) is a promoter which controls expression of the heterologous gene(s). The promoter is preferably the mouse cytomegalovirus (CMV) early promoter, or an effective expression promoting fragment thereof. Positioned upstream of the promoter, is the left end replication and packaging elements of the adenovirus-5 genome. A eukaryotic splice acceptor and splice donor site is positioned immediately downstream of the promoter.
Following the splicing sequence elements, is the gene insertion site(s), which is followed by the polyadenylation sequence, and the region for homologous recombination which contains a portion of the adenovirus-5 genome. The polyadenylation sequence preferably comprises the 3xe2x80x2 processing site taken from the mouse xcex2-globin transcription unit i.e., Globin poly(A). The order and choice of the splicing and polyadenylation elements results in optimal processing of the pre-mRNA into mRNA. The region for homologous recombination preferably is the adenovirus-5 genome nucleotide sequence 2800-5776.
The plasmid vector of the invention can be readily converted into a recombinant adenovirus for expression of a heterologous gene(s) and/or gene product(s) in a mammalian cell. Here, the plasmid vector is co-transfected with the large fragment of the adenovirus-5 genome i.e., 3.8-100 map units and/or an appropriate derivative thereof. Homologous recombination between these DNA fragments results in the production of a replication defective, recombinant adenovirus. The recombination reconstructs the adenovirus-5 genome by displacing the E1A and E1B protein coding regions with the plasmid vector cDNA.
In another embodiment of the invention, there is provided a recombinant adenovirus expression system for the receptor for thyrotropin-releasing hormone (TRH-R). The recombinant adenovirus, designated AdCMVmTRHR, circumvents difficulties encountered when using conventional transient or stable plasmid expression systems. Using this recombinant adenovirus (AdCMVmTRHR), TRH-Rs can be expressed in different mammalian cell types, including those resistant to transient transfection assay. Recombinant adenovirus, AdCMVmTRHR, was produced by homologous recombination between plasmid vector, designated pAdCMVmTRHR, i.e. the generic plasmid vector of the invention containing the gene coding TRH-R, co-transfected with the large fragment of adenovirus-5 d1309 genome. The versatility of using adenovirus mediated gene transfer and expression of TRH-Rs not only facilitates in vitro studies of TRH-R biology, but provides a valuable in vivo expression vector capable of extending TRH-R studies in animal model systems.
In a further embodiment of the present invention, infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo was characterized using the recombinant adenovirus of the invention. The recombinant adenovirus, designated AdCMVCATgD, includes the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus (CMV) promoter. Plasmid vector pAdCMVCATgD i.e., generic plasmid vector of the present invention containing the gene encoding the bacterial CAT sequence, was co-transfected with the large fragment of the adenovirus-5 genome (3.6-100 map units). Homologous recombination between the plasmid vector and adenovirus fragment produced the recombinant adenovirus, designated AdCMVCATgD.
Virtually all fetal or adult cardiocytes expressed the CAT gene in vitro when infected with 1 plaque forming unit (pfu) of virus per cell. Using in vitro studies as a guide, recombinant virus AdCMVCATgD was introduced directly into adult rat myocardium and the expression results obtained from virus injection was compared to those obtained by direct injection of plasmid vector pAdCMVCATgD DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium is orders of magnitude higher than that seen from DNA injection and is proportional to the amount of input virus. The recombinant adenovirus-mediated gene delivery system is a very effective tool for high efficiency gene transfer into the cardiovascular system.